This study reports the reconstitution of the de novo activation pathway of procaspase-9 using highly purified cytochrome c, recombinant APAF-1, and recombinant procaspase-9. APAF-1 binds and hydrolyzes ATP or dATP to ADP or dADP, respectively. The hydrolysis of ATP/dATP and the binding of cytochrome c promote APAF-1 oligomerization, forming a large multimeric APAF-1-cytochrome c complex. This complex can be isolated using gel filtration chromatography and is sufficient to recruit and activate procaspase-9. The stoichiometric ratio of procaspase-9 to APAF-1 is approximately 1:1 in the complex. Once activated, caspase-9 disassociates from the complex and becomes available to cleave and activate downstream caspases such as caspase-3. Caspases are key enzymes in apoptosis, causing morphological changes such as nuclear membrane breakdown, chromatin condensation and fragmentation, cell membrane blebbing, and formation of apoptotic bodies. Caspases cleave various cellular substrates, leading to apoptosis. Caspases involved in apoptosis are present as inactive zymogens in living cells and become activated through intracellular caspase cascades. Two well-characterized caspase cascades are described: one initiated by cell-surface death receptors, and the other triggered by cytochrome c released from mitochondria, which activates caspase-9 through APAF-1. APAF-1 is a 130-kDa protein consisting of a CED-4 homologous domain flanked by a caspase recruitment domain (CARD) and 12 WD-40 repeats. APAF-1 is essential for apoptosis, as shown by knock-out experiments in mice. Animals lacking the Apaf-1 gene show defects in apoptosis, including excessive neurons in the brain, facial abnormalities, and delayed recession of interdigital webbing. Cells derived from these animals show resistance to various apoptotic stimuli. The biochemical mechanism of procaspase-9 activation by APAF-1 is not well understood. Previous studies have shown that APAF-1 forms a complex with caspase-9 in the presence of dATP and cytochrome c. APAF-1 truncated at the COOH-terminal WD-40 repeats is constitutively active in vitro, independent of cytochrome c and dATP. Truncated APAF-1 interacts with itself, suggesting that oligomerization is important for its function. However, there is no direct evidence for APAF-1 oligomerization or the roles of dATP/ATP and cytochrome c in this process. In this study, the procaspase-9 activation pathway was reconstituted using highly purified cytochrome c,This study reports the reconstitution of the de novo activation pathway of procaspase-9 using highly purified cytochrome c, recombinant APAF-1, and recombinant procaspase-9. APAF-1 binds and hydrolyzes ATP or dATP to ADP or dADP, respectively. The hydrolysis of ATP/dATP and the binding of cytochrome c promote APAF-1 oligomerization, forming a large multimeric APAF-1-cytochrome c complex. This complex can be isolated using gel filtration chromatography and is sufficient to recruit and activate procaspase-9. The stoichiometric ratio of procaspase-9 to APAF-1 is approximately 1:1 in the complex. Once activated, caspase-9 disassociates from the complex and becomes available to cleave and activate downstream caspases such as caspase-3. Caspases are key enzymes in apoptosis, causing morphological changes such as nuclear membrane breakdown, chromatin condensation and fragmentation, cell membrane blebbing, and formation of apoptotic bodies. Caspases cleave various cellular substrates, leading to apoptosis. Caspases involved in apoptosis are present as inactive zymogens in living cells and become activated through intracellular caspase cascades. Two well-characterized caspase cascades are described: one initiated by cell-surface death receptors, and the other triggered by cytochrome c released from mitochondria, which activates caspase-9 through APAF-1. APAF-1 is a 130-kDa protein consisting of a CED-4 homologous domain flanked by a caspase recruitment domain (CARD) and 12 WD-40 repeats. APAF-1 is essential for apoptosis, as shown by knock-out experiments in mice. Animals lacking the Apaf-1 gene show defects in apoptosis, including excessive neurons in the brain, facial abnormalities, and delayed recession of interdigital webbing. Cells derived from these animals show resistance to various apoptotic stimuli. The biochemical mechanism of procaspase-9 activation by APAF-1 is not well understood. Previous studies have shown that APAF-1 forms a complex with caspase-9 in the presence of dATP and cytochrome c. APAF-1 truncated at the COOH-terminal WD-40 repeats is constitutively active in vitro, independent of cytochrome c and dATP. Truncated APAF-1 interacts with itself, suggesting that oligomerization is important for its function. However, there is no direct evidence for APAF-1 oligomerization or the roles of dATP/ATP and cytochrome c in this process. In this study, the procaspase-9 activation pathway was reconstituted using highly purified cytochrome c,